Prostaglandin E₂ (PGE₂) is one major prostanoid produced under inflammatory situation. Although PGE₂ is known to induce vascular contraction, its detailed mechanism remains unknown. In the present study, we investigated the signaling pathway underlying PGE₂-induced smooth muscle contraction in rat mesenteric artery. PGE₂ (0.3–30 μM) concentration-dependently caused contraction in endothelium-denuded artery. RT-PCR showed that this artery expresses mRNAs for all four prostanoid EP receptors (prostanoid EP_1–4). Among selective agonists for PGE₂ receptors, only a prostanoid EP₃ receptor agonist, ONO-AE-248 (0.3–30 μM) induced contraction. Consistently, pretreatment with a prostanoid EP₃ antagonist (L-798106, 1 μM) significantly but not completely inhibited the PGE₂-induced contraction. Interestingly, pretreatment with a prostanoid FP antagonist (AL8810, 1 μM) or a TP antagonist (SQ29548, 10 nM) also partially inhibited the PGE₂-induced contraction. Since ONO-AE-248 (10 μM) did not influence intracellular Ca²⁺ concentration in mesenteric artery, we next examined the involvement of Ca²⁺-independent contractile pathway including PKCs and ROCK in prostanoid EP₃-mediated contraction. Pretreatment with bisindolyl-maleimide I (a general PKC inhibitor, 1 μM), Ro-31-8425 (a conventional PKC and PKCε inhibitor, 1 μM), rottlerin (a selective PKCδ inhibitor, 1 μM) and Y-27632 (a ROCK inhibitor, 1 μM) but not Go 6976 (a conventional PKC inhibitor, 1 μM) attenuated 10 μM ONO-AE-248-induced vascular contraction. In western blot analysis, we confirmed that the treatment with ONO-AE-248 (10 μM, 30 min) phosphorylated PKCδ (Thr505) and PKCε (Ser729). These results suggest that PGE₂ induces vascular smooth muscle contraction via prostanoid EP₃, FP and TP receptors in rat mesenteric artery. Prostanoid EP₃-mediated contraction is ascribed to Ca²⁺-independent contractile pathway including PKCδ, ε and ROCK.